ABSTRACT
Objective:To study the effect of the siderophore virulence gene entB on the virulence of carbapenem-resistant Klebsiella pneumonia (CRKP). Methods:CRKP-27 was selected as the experimental strain from 30 CRKP strains collected from the First Affiliated Hospital of Kunming Medical University. The knockdown strain (Δ entB) and complementing strain (C-Δ entB) were constructed by the clustered regularly interspaced short palindromic repeat-Cas9 technology, and verified by polymerase chain reaction (PCR). In order to initially understand the effect of entB on CRKP colony morphology and virulence phenotype, the colony morphology of CRKP-27, Δ entB, and C-Δ entB strains were observed and string test were tested. Draw the growth curve of the strains and determine the effect of entB on the growth of the CRKP strains. The siderophores production ability of the strains were detected quantitatively using chrome azurol S (CAS) detection solution. Mice model of inflammation was established to observe the survival rate of mice and intuitively understand the effect of entB on CRKP virulence. Results:The PCR results showed that the Δ entB strain and C-Δ entB stranin were constructed successfully. The entB has no significant effect on the colony morphology, capsule and virulence phenotype of CRKP. The growth rate of Δ entB was significantly faster than that of CRKP-27( P=0.008) and C-Δ entB ( P=0.001), which showed that entB weakened the growth ability of CRKP. Compared with CRKP-27( P=0.001) and C-Δ entB( P=0.001), the siderophore production of Δ entB was significantly decreased by 11.739 3% and 11.964 2%, indicating that entB gene increased the capacity of CRKP to produce siderophpres. In animal experiments, compared with CRKP-27( P=0.023) and C-Δ entB( P=0.024), the survival rate of mice in the Δ entB group was significantly increased, indicating that the entB increased the virulence of the CRKP. Conclusion:The siderophore virulence gene entB significantly weakened the growth ability of the strain, but clearly enhanced the siderophore production capacity and virulence of CRKP.
ABSTRACT
Objective:To investigate the mechanism by which Fusobacterium nucleatum ( Fn) infection promotes TNF-α-induced inflammatory changes in colorectal cancer HCT116 cells. Methods:Fn-infected cells and TNF-α inflammation induction models were established and divided into 4 groups, namely uninfected control group, Fn-infected group, TNF-α induction group, and Fn+ TNF-α group. First, Fn was used to infect normal colonic epithelial cells hcoEPIC, colorectal cancer HCT116 and LoVo cells, the cell adhesion was detected 4 h later. Subsequently, HCT116 cells were induced with TNF-α for 3 h and then infected with Fn. After 24 h, the cell survival rate and cell damage were detected by CCK8 experiment and lactate dehydrogenase (LDH) viability assay. The ELISA method was further used to detect the expression of nuclear transcription factor NF-κB and cytokines IL-6, IL-8, and IL-1β in the cell and cell culture supernatant. Results:Fn has strong adhesion to colorectal cancer cells HCT116 and LoVo ( P<0.05), but basically does not show invasion. On the contrary, it has a higher invasion rate to normal colonic epithelial cells hcoEPIC after 24 h. Compared with the uninfected Fn group, the cell survival rate of the Fn-infected group was significantly reduced and the cell damage increased ( P<0.001). Three hours after TNF-α induction, Fn infection further promoted cell death and damage ( P<0.001). The expression of NF-κB in the Fn infection and TNF-α alone treatment group was significantly higher than that of the uninfected group ( P<0.001, P<0.05), and the NF-κB expression in the Fn+ TNF-α group was significantly higher than that of the control group and the single treatment group ( P<0.001). In the Fn infection and TNF-α treatment groups, the expressions of IL-6 and IL-8 were significantly higher than those in the uninfected group ( P<0.001), and IL-1β did not change significantly ( P>0.05). The expressions of IL-6, IL-8 and IL-1β in the Fn+ TNF-α group were significantly higher than those in the normal control group and the single treatment group ( P<0.05). Conclusions:Fusobacterium nucleatum can preferentially adhere to colorectal cancer cell HCT116, further promote TNF-α-induced cell damage and death, the expression and release of NF-κB and its downstream pro-inflammatory cytokines IL-6, IL-8, IL-1β.